1. Field of the Invention
This invention relates to antithrombotic agents and uses thereof. Specifically, the invention relates to compounds that bind to platelets, specifically compounds that bind to the platelet receptor molecule GPIIb/IIIa. Such compounds include peptides and cyclic peptides that bind to platelets and inhibit their aggregation. In particular, the platelet-specific antithrombotic agents of the invention are covalently linked to a polyvalent linker moiety, so that the polyvalent linker moiety is covalently linked to a multiplicity of the platelet-specific moieties, particularly peptides and cyclic peptides, thereby providing the multimeric polyvalent antithrombotic agents of the invention. The invention also provides methods for using such compounds to prevent the formation of thrombi at sites in a mammalian body.
2. Description of the Prior Art
Thrombosis and thromboembolism, in particular deep vein thrombosis (DVT) and pulmonary embolism (PE), are common clinical conditions that are associated with significant morbidity and mortality. It has been estimated that in the U.S. approximately 5 million patients experience one or more episodes of DVT per year and that over 500,000 cases of pulmonary embolism occur, resulting in 100,000 deaths (J. Seabold, Society of Nuclear Medicine Annual Meeting 1990).
In addition, myocardial infarction (heart attack) is usually caused by thrombosis in a coronary artery, often at the site of an atherosclerotic plaque. Conventional therapies for heart attack involve removing such thrombi, either surgically by angioplasty and/or by adminstration of thrombolytic drugs, such as recombinant tissue plasminogen activator or streptokinase. Following such therapy, however, re-stenosis or even re-occlusion of the affected coronary artery frequently occurs due to formation of another thrombus at the site of the original thrombus. Preventing such re-occurrence of coronary artery thrombi is thus an important goal of all post-infarct therapy.
The physiological processes involved in the formation of thrombi are initiated by the accumulation of platelets at sites of damage or insult to the endothelial cell wall of a blood vessel, such as an atheroschlerotic plaque. Platelets normally accumulate at such sites after stimulation by local mediators signalling the injury. Subsequently, such platelets become aggregated at such sites by binding serum fibrinogen via GPIIb/IIIa receptors expressed on the platelet surface. It is with the binding of fibrinogen that such an aggregation of platelets becomes a thrombus.
The amino acid sequence of the fibrinogen molecule recognized by GPIIb/IIIa receptors is the sequence -Arg-Gly-Asp- (RGD), which sequence is present four times in each fibrinogen molecule. Platelet aggregation can be inhibited using fibrinogen antagonists that bind to the GPIIb/IIIa receptors. Thus, compounds that are fibrinogen antagonists and bind to GPIIb/IIIa are useful in preventing thrombosis, particularly in post-angioplasty or post-thrombolytic treatment regimes.
Peptides having the ability to bind to platelets and inhibit their aggregation are known in the prior art.
Ruoslahti & Pierschbacher, U.S. Pat. No. 4,578,079 describe peptides of sequence X-Arg-Gly-Asp-R-Y, wherein X and Y are either H or an amino acid, and R is Thr or Cys, the peptides being capable of binding to platelets.
Ruoslahti & Pierschbacher, U.S. Pat. No. 4,792,525 describe peptides of sequence Arg-Gly-Asp-X, wherein X is Ser, Thr or Cys, the peptides being capable of binding to platelets.
Pierschbacher er al., 1989, International Application No. WO089/05150 disclose conformationally-restricted RGD-containing peptides for inhibiting cell attachment to a substratum.
Hawiger et al., 1989, International Application No. WO089/10135 relates to peptides comprising sequences for two binding sites of a protein.
Nutt et al., 1990, European Patent Application, Publication No. 0410537A1 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application, Publication No. 0410539A1 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application, Publication No. 0410540A1 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application, Publication No. 0410541A1 disclose cyclic RGD peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application, Publication No. 0422937A1 disclose cyclic peptides that are fibrinogen receptor antagonists.
Nutt et al., 1990, European Patent Application, Publication No. 0422938A1 disclose cyclic peptides that are fibrinogen receptor antagonists.
Ali et al., 1990, European Patent Application, Publication No. 0425212A2 disclose cyclic peptides that are fibrinogen receptor antagonists.
Barker et al., 1991, International Application No. WO91/01331 disclose cyclic peptides for inhibiting platelet aggregation.
Maraganore et al., 1991, International Application No. WO91/02750 disclose a radiolabeled thrombus inhibitor comprising (a) an inhibitor moiety; (b) a linker moiety; and (c) and an "anion binding exosite (ABE)" binding site moiety.
Pierschbacher et al., 1991, International Application No. WO91/15515 disclose cyclic peptides that are fibrinogen receptor antagonists.
Ojima et al., 1992, 204th Meeting, Amer. Chem. Soc. Abst. 44 disclose synthetic multimeric RDGF peptides useful in inhibiting platelet aggregation.
Although it is possible to prepare cyclic peptides that specifically bind to platelets, some such peptides exhibit low binding site affinity whereby the strength of peptide binding to platelets is insufficient to prevent platelet aggregation and thereby have an antithrombotic effect. Peptides comprised of linear arrays of thrombus-specific peptide binding units have been described in the prior art.
Rodwell et al., 1991, International Application No. WO91/17173 disclose linear arrays of the peptide sequence RGD.
Kitaguchi et al., European Patent Application, Publication No. 0503301A2 disclose peptides derivatives of Arg-X-Asp peptides having anti-metastatic properties.
Pierschbacher et al., International Patent Application No. WO90/06767 disclose peptide-polymer conjugates between RGD and (dR)GD peptides and biodegradable polymers.
Kojima et al., European Patent Application, Publication No. 0482649A2 disclose CM-chitin derivatized with RGD-containing peptides.
Ahlem et al., European Patent Application, Publication No. 0446071A2 disclose tris-maleimido compounds for crosslinking antibodies and antibody fragments.
Cheronis et al., 1992, J. Med. Chem. 35: 1563-1572 disclose chemical crosslinking of bradykinin.
Japanese Patent Application, Publication No. JP1309682 disclose the use of polysaccharide-RGD conjugates for promoting cell adhesion in vitro.
Alternative arrangements of specific binding peptide units are preferable. The present invention provides multimeric polyvalent antithrombotic agents, one embodiment of which are reagent comprised of a multiplicity of cyclic peptides that specifically bind to platelets and have a sufficient affinity for platelets to prevent their aggregation. The incorporation of a multiplicity of platelet-specific cyclic peptides in the antithrombotic agents of the invention permits the use of particular platelet-specific cyclic peptides comprising platelet binding sequences whose individual binding affinity might not otherwise be sufficient to produce the desired inhibition of platelet aggregation resulting in an antithrombotic effect in vivo, but which have other desireable properties, such as improved in vivo stability and half-life, which are evidenced for example by increased retention times at thrombus sites in vivo, detected by Tc-99m scintigraphy. Improved inhibition of platelet aggregation by particular platelet-specific binding moieties including peptides and cyclic peptides is achieved using the multimeric polyvalent antithrombotic agents of this invention.